▉ Introduction
Antibody-drug conjugates (ADCs) are currently developing rapidly, utilizing antibodies to selectively deliver cytotoxic drugs to tumor sites. As of May 2021, the U.S. Food and Drug Administration (FDA) has approved 10 ADCs, including Adcetris®, Kadcyla®, Besponsa®, Mylotarg®, Polivy®, Padcev®, Enhertu®, Trodvy®, Blenrep®, and Zynlonta™ for use as monotherapy or in combination for the treatment of breast cancer, urothelial carcinoma, multiple myeloma, acute leukemia, and lymphoma. By July 2021, there were 13 ADC drugs on the market globally, with over 80 ADCs under investigation in approximately 150 active clinical trials.
Despite the growing popularity of ADCs, expanding their therapeutic index (better efficacy, less toxicity) remains a challenge. However, the development of certain new technologies, such as site-specific conjugation, linkers, payloads, new biological platforms, and advanced analytical techniques, is helping to shape the future of ADC development.
(🔗Review of ADC)
1. Mechanism of Action and Key Elements of ADCs
Most ADCs follow a similar mode of action, including antibody-mediated receptor binding, internalization of the ADC, followed by the release of the active payload and cytotoxicity (see figure below), and of course, the bystander effect. The success of ADCs depends on several key factors.
1) Target antigen (e.g., CD30, HER2, CD22, CD33, CD79b, Nectin 4, Trop2, BCMA, CD19)
2) Type of antibody (e.g., IgG1, IgG2, IgG4, nanobodies, bispecific antibodies).
3) Type of payload (e.g., MMAE, DM4, calicheamicin, DM1, MMAF)
4) Type of linker (e.g., valine-citrulline, Sulfo-SPDB, hydrazone linkers),
5) Linker platform (e.g., lysine, cysteine, and site-specific conjugation).
6) Target indications (e.g., breast cancer, lymphoma, leukemia, uroepithelial cancer, lung cancer, ovarian cancer, etc.).
（🔗Review of ADC Mechanism of Action；🔗ADC Linker Overview）
Mechanism of Action of ADCs
2. ADC Drugs in Clinical Use
Currently, there are over 80 ADCs undergoing active clinical trials, with most in Phase I and I/II trials (see figure a and table at the end). More than 80% of clinical trials are investigating the safety and efficacy of ADCs in various solid tumors, while the remaining trials involve hematologic malignancies (see figure b). This indicates that, following the early success of T-DM1 and the recent approvals of T-Dxd, sacituzumab govitecan, and enfortumab vedotin, the trend toward studying ADCs in solid tumors has been gradually increasing in recent years. The ADCs in these 80+ clinical trials target approximately 40 different targets (see figure c).
Currently, HER2 is one of the most attractive targets in ADC drug development, with three anti-HER2 ADCs currently in Phase III trials. Among them, RC48 produced by RemeGen, links the anti-HER2 IgG1 antibody hertuzumab with approximately four MMAE molecules via a valine-citrulline linker that can be cleaved by proteases. In preclinical studies, lower doses of RC48 showed anti-tumor activity in xenograft models sensitive and resistant to trastuzumab and lapatinib. It demonstrated better anti-tumor efficacy compared to T-DM1. Studies have shown that in multiple Phase I trials targeting HER2-positive malignancies, its safety can be effectively controlled, and in a Phase II pivotal trial (NCT03507166), it achieved encouraging results: including a total response rate (ORR) of 51.2% in pre-treated HER-2 positive locally advanced or metastatic urothelial carcinoma.
ADC Drugs for Tumor Treatment in Clinical Use
In the field of small molecule drugs, most disrupt microtubule proteins and cause mitotic arrest, while a few induce DNA damage (see figure 3d). Of course, some new small molecule drugs are gradually being used in ADCs, such as TRL7/8 (Toll-like receptor agonists), RNA polymerase II inhibitors, and BCL-xL anti-apoptotic protein targeting.
Conjugation Methods can directly affect the quality of ADCs. The quality of ADCs in turn affects the safety and efficacy of the product. Currently, there are three main conjugation methods: through reduced interchain disulfide bonds in cysteines, lysines exposed on the surface of antibodies, and site-specific conjugation techniques. Most ADCs in clinical trials are either traditionally cysteine-conjugated or site-specifically conjugated under manufacturer licenses. Only a small portion use traditional lysine conjugation methods, which may lead to significant heterogeneity in ADC drugs (see figure e).
3. Challenges in ADC Development
There are many challenges that need to be faced in the development of ADC drugs. The first is to demonstrate the efficacy of the drug, as many drugs have been forced to terminate due to the inability to prove their efficacy in clinical settings, such as MM-302 and Rova-T.
In addition, the toxicity associated with ADC drugs is also a challenge. For example, the use of calicheamicin as a payload is associated with an increased incidence of liver damage and hepatotoxicity. This includes an increase in the incidence of veno-occlusive disease (also known as sinusoidal obstruction syndrome) and drug-induced liver damage. During the clinical trial and post-approval use of gilteritinib, an increase in the incidence of veno-occlusive disease (also known as sinusoidal obstruction syndrome) and drug-induced liver damage was observed. Additionally, there are reports of side effects associated with MMAE causing peripheral neuropathy and neutropenia, and MMAF causing ocular toxicities, as well as topoisomerase I inhibitors causing neutropenia.
Effective payload MMAE (https://www.adcreview.com/adc-university/adcs-101/targeting-cancer-adcs/)
Development of Resistance: Over time, tumors can develop mechanisms to overcome drug efficacy, leading to weakened or lost efficacy. Because ADCs exert their effects through multiple pathways, resistance can occur at any step of the drug’s action (see figure).
One mechanism of resistance may stem from regulation of antibody recognition of antigens. This may be due to downregulation of expression of the target on the cell surface. Some preclinical studies have shown that cells continuously treated with ADCs eventually develop acquired resistance models, with reduced expression of target antigen proteins. To address such resistance mechanisms, some dual-targeting bispecific antibody ADC drugs are already in clinical development, such as ZW49 (targeting different epitopes of Her2) and M1231 (targeting EGFR and UC1).
Another common mechanism of resistance is through ATP-binding cassette transporters clearing the effective payload. Many cytotoxic payloads used in ADCs may be substrates of these pumps, which can lead to drug efflux from target cells and reduced efficacy. Clinical data indicate that efflux pumps are one of the reasons for the reduced efficacy of gilteritinib.
Of course, any step in the action of ADC drugs can potentially produce resistance: 1) defects in antibody internalization, transport, and recycling, 2) lysosomal degradation leading to drug release barriers, and 3) changes in cell death pathways (see figure).
Mechanisms of Resistance in ADCs
4. Important Considerations in ADC Design
4.1 Target Antigen
Improving the safety and efficacy of ADCs largely depends on the selection of target antigens and their interactions. Two key parameters are involved in the selection of target antigens: tumor specificity and expression level. Ideally, the selected target will exhibit high tumor-specific or disease-specific expression, and the specificity of the target with minimal or no expression in normal tissues is crucial for reducing the toxicity of ADCs, thus playing an important role in the overall success of ADCs.
From an oncology perspective, antigens can be expressed as surface receptors on tumor cells, tumor stem cells, or in tumor vasculature and microenvironments. In the best case, the antigen is uniformly expressed at similar levels across all tumor-associated cells. This way, ADC drugs can fully utilize the bystander effect to overcome tumor heterogeneity.
4.2 Monoclonal Antibodies
After selecting target antigens, antibodies need to be screened based on their tumor penetration capabilities and antibody subtypes. ADCs currently under development and approval belong to IgG1, IgG2, or IgG4 subclasses. These subclasses differ in crosslinking capabilities and biological activity, including ADCC and complement-dependent cytotoxicity (CDC).
Compared to IgG2 and IgG4, IgG1 is the most commonly used because it has stronger delivery capabilities and more effector functions. However, when considering target characteristics or different mechanisms, in some cases, the ADCC and CDC effects of IgG1 need to be avoided, making IgG2 and IgG4 antibodies the preferred choice. Additionally, subtype selection greatly impacts conjugation, especially when considering the use of cysteine for conjugation.
Structures of IgG1, IgG2, or IgG4 Antibodies
4.2.1 Size of Monoclonal Antibodies
After selecting antigens and antibody subtypes, the size of the antibody needs to be considered. Historically, full-length IgG antibodies were often chosen, but these antibodies have certain limitations in cellular internalization and tumor penetration. To address this issue, some new forms of antibodies have emerged, such as Fab-ADC, ScFv-ADC, etc. These smaller antibodies have better permeability, but because they lack Fc, their half-lives are relatively short.
4.2.2 Antibody Modifications
Like other proteins, antibodies can undergo modifications during production or storage (PTMs), such as deamination, sialylation, or cleavage of C-terminal lysines, which can affect the stability of the antibody, and thus affect the stability of ADCs, impacting their efficacy.
4.2.3 Internalization of ADCs
Most ADCs are designed against target antigens, which show efficient internalization via receptor-mediated endocytosis to facilitate ADC entry into cells after recognition. For a long time, receptor internalization has been a requirement for effective ADC design to minimize the impact of cytotoxic payload release on healthy cells.
To design a successful internalizing ADC, it is essential to evaluate the accessibility, density, internalization rate, and intracellular transport of the ADC’s target. Generally speaking, compared to hematologic malignancies, ADCs targeting antigens expressed on solid tumors face more physical barriers that must be overcome to reach the antigens after administration. In hematologic malignancies, the targets are easily exposed to circulating ADCs. Furthermore, targets can sometimes